This invention is related to a novel process for preparing antibacterial .beta.-lactams i.e. penicillins and cephalosporins by the action of an acylase on a penicillin or cephalosporin nucleus amine as substrate and an ester (I) of the following formula as the acyl source: ##STR2## (wherein RCO is an acyl group in penicillin or cephalosporin side chains;
X is a hydrogen atom, lower alkyl group or hydroxy-lower alkyl group; PA1 Y is a hydrogen atom or a lower alkyl group; and n is a positive integer) PA1 a. Meat extract agar plate culture (28.degree. C., 2 days) Pinkish circular colonies with shining surface are formed. They are entire, convex and opaque colonies with butyrous structure. No soluble pigment is observed. PA1 b. Meat extract agar slant culture (28.degree. C., 1 day) Moderate pinkish and filamentous growth is observed. The colonies are convex and opaque wih a shining surface. No soluble pigment is observed. PA1 c. Meat extract liquid culture (28.degree. C., 2 days) Filamentous growth is observed on the surface with homogeneous turbidity and some precipitation. PA1 d. Meat extract gelatin stub culture (24.degree. C. 30 days) Growth is observed on the surface but no gelatin liquefaction. PA1 e. Litmus milk culture (28.degree. C., 7 days) A little decolorization occurs at neutral pH and coagulation and peptonization of milk are not observed. PA1 a. Meat agar plate culture (28.degree. C., 2 days) Pale yellowish circular colonies with a shining surface are formed. They are entire, convex and opaque colonies having butyrous structure. No soluble pigment is observed. PA1 b. Meat extract agar slant culture (28.degree. C., 1 day) Moderate, yellowish and filamentous growth is observed. The colonies are convex and opaque with a shining surface. No soluble pigment is observed. PA1 c. Meat extract liquid culture (28.degree. C., 2 days) Homogenous turbidity but no growth is observed with some precipitation. PA1 d. Meat extract gelatin stub culture (24.degree. C., 30 days) Growth on surface is observed but no gelatin liquefaction. PA1 e. Litmus milk culture (28.degree. C., 7 days) Decolorization at alkaline pH and no coagulation are observed with gradual peptonization. PA1 a. Meat agar plate culture (28.degree. C., 2 days) Cream-colored circular colonies with a shining surface are formed having entire, convex, opaque and butyrous structure. No soluble pigment is observed. PA1 b. Meat extract agar medium (28.degree. C., 1 day) Moderate, cream-colored and filamentous growth is observed. Colonies are opaque with shining surface. PA1 c. Meat extract liquid culture (28.degree. C., 2 days) No growth on surface but homogenous turbidity is observed. PA1 d. Meat extract gelatine stub culture (24.degree. C., 30 days) Growth on surface which extends into the inside is observed. Liquefaction proceeds gradually after incubation for 10 days. PA1 e. Litmus milk culture (28.degree. C., 7 days) No change occurs at neutral pH and acidification begins on about the 22nd day with gradual coagulation. PA1 f. Sabouraud-dextrose agar slant culture (28.degree. C., 2 days) Little growth is observed. PA1 g. Soybean agar slant culture (28.degree. C., 2 days) Moderate growth is observed. PA1 h. Tyrosine agar plate culture (28.degree. C.) Moderate growth is observed. The medium does not turn brown.
The reaction for the synthesis proceeds in a homogeneous solution and is suitable for efficient continuous production. This overcomes previous troubles preventing commercial acylation for production of penicillins and cephalosporins by the action of an acylase.
Processes are well known for preparing penicillins or cephalosporins by reacting a methyl or ethyl ester of an acid having acyl moiety to be introduced with 6-aminopenicillanic acid or 7-aminocephalosporanic acid or reactive derivatives thereof (e.g. Japanese Patent Application Publication No. 47-25388, 47-29588, 48-26985, 48-35090, 48-99393, 49-14687, 49-36890, 49-48892, 49-75787, 49-134893 and 52-110896). It is to be noted that the acyl source of above references are invariably a lower alkyl ester, especially methyl ester.
The present inventors observed during similar reactions that many lower alkyl esters are sparingly soluble in water and attain only low concentration insufficient for effective acylation.
As a result, known synthesis with immobilized enzyme often has difficulty due to two-phase formation or plug formation in a column to prevent smooth elution.
To solve these problems, many esters are made and tried on substrates for the acylases to find (poly)ethylene glycol ester and the like very suitable. These esters are freely miscible with water and do not form plugs in a column upon acylation. Therefore they can be effectively used in continuous reactions with a column of immobilized enzyme. Thus, the molar ratio of acyl source and amino source could be reduced, yield could be improved, concentration of substrates and products could be elevated, unreacted starting material and products could be collected from a reaction mixture efficiently and other merits have been discovered for enzymatic synthesis.
Thus, this invention is a novel enzymatic synthesis of .beta.-lactam antibacterials by reacting an ester of the formula (I) with aminoazetidinone carboxylic acid of the formula (II) in the presence of acylase in an aqueous medium to produce a .beta.-lactam antibacterial of the formula (III):
______________________________________ ##STR3## (I) [acyl source] H.sub.2 NQ (II) [amino source] RCONHQ (III) [.beta.-lactam ##STR4## antibacterial] ______________________________________
[wherein
RCO-- is an acyl group;
X-- is a hydrogen atom, lower alkyl group or hydroxy-lower alkyl group;
Y-- is a hydrogen atom or a lower alkyl group;
n is a positive integer; and
Q-- is a group of the formula: ##STR5##
(in which Z is a hydrogen or halogen atom, a nucleophilic group, a methyl, halomethyl or methyl substituted by a nucleophilic group)]
As will be explained later, enzymes of bacterial or fungal origin are especially important as acylase for this invention from the view point of e.g. production, efficiency, cost and stability.
Especially suitable bacteria include acylase-producing bacteria belonging e.g. to genus Micrococcus, Arthrobacter or Bacillus, including specific strains of Micrococcus roseus M-1054-1 (FERM-P 3744), Micrococcus luteus M-331-1, Arthrobacter globiformis M-345-2 (FERM-P 3743), Bacillus circulans M-1123-5 (FERM-P 5153) and Bacillus megaterium NRRL B-5385.
The compounds of formula (I) are novel compounds indispensable as constituents of this invention and are highly water-soluble concomitantly susceptible as substrate of the acylases. They are useful acyl sources and are useful from this point of view.
In the above formula, the acyl group represented by RCO-- is an acyl of natural or synthetic, penicillins or cephalosporins, which is susceptible as a substrate of the said amido-acylase in a form of the ester derivative (I).
Representative RCO-- groups can be straight, branched, cyclic or partially cyclic lower alkanoyl or lower alkenoyl; monocyclic lower aralkanoyl, monocyclic aryloxylower alkanoyl, (O, N or S)-heterocyclic-lower alkanoyl, (O, N or S)-heterocyclic thio-lower alkanoyl, cyanoacetyl, cyanomethylthioacetyl, monocyclic arylglycyl, monocyclic cycloalkenylglycyl, monocyclic arylglycolyl, N-acyl-arylglycyl, monocyclic arylmalonyl or arylsulfoalkanoyl, all above optionally having lower alkyl, aminomethyl, halogen, hydroxy, lower alkanoyloxy or lower alkoxy as a substituent, and preferably containing 1 to 15 carbon atoms.
Said ester (I) has as an alcoholic moiety containing n units of an ethyleneglycol unit of the formula --(CH.sub.2 CHXO)-- as a constituent. Said number n is usually from 1 to 20, especially from 4 to 15. Another end Y of the ethyleneglycol chain can be a hydrogen or in the form of a lower alkyl ether. It has been found that enzymatic activity for the acyl source is not greatly affected by the ethyleneglycol chain length. As a result, the ester (I) used as acyl source can be a mixture of several esters having various n values to obtain equivalent effects.
These esters can be prepared by a conventional method e.g. by the reactions of an acid RCOOH with glycerin or (poly)ethyleneglycol or ethylene oxide, by dehydration, condensation, or through acid halide, or chemically reactive esters of another type for ester exchange reaction.
The aminoazetidinone carboxylic acid (II) is a penicillin nucleus amine or cephalosporin nucleus amine capable of being used as a substrate of the acylase applicable in a form of water soluble salt or ester.
In the formula (II), Q is penicillin or cephalosporin nucleus represented by the following partial formula: ##STR6## (wherein Z is as defined above).
Said group Z is a substituent at the 3-position of cephalosporin antibacterials i.e. hydrogen or halogen atom, or nucleophilic group or methyl optionally substituted by said nucleophilic group. Such nucleophilic group has been disclosed in e.g. Japanese Patent Application Publication No. 49-81381.
Representative Z groups may have up to 7 carbon atoms including hydrogen, halogen, lower alkoxy, monocyclic (O, N or S)-heterocyclic thio, lower alkyl, monocyclic lower aralkyl, lower alkanoyloxy, lower alkanoyloxy-lower alkyl, monocyclic (O, N or S)-heterocyclic thio-lower alkyl and pyridinium-lower alkyl. These can be substituted by lower alkyl, carboxy-lower alkyl, lower alkoxy, carbamoyl or halogen. A heterocycle in RCO or Z has 1 to 4 heteroatoms.
An aminoazetidinone carboxylic acid (II) can be used in a form of enzymatically acceptable water-soluble salts (e.g. sodium salt, potassium salt, calcium salt) or water-soluble ester (e.g. methanesulfonylethyl ester, acetonyl ester, lower alkoxy-lower alkyl ester), or it may be used in a form of water-soluble acid addition salt (e.g. mineral acid salt, carboxylic acid salt or sulfonic acid salt) as an equivalent starting material. It is a known compound or closely related compound which can be produced from known substance with a known a method.
The enzyme for this invention can be an acylase of plant, animal, fungal or bacterial origin. Among them, the most suitable industrially are fungal or bacterial enzyme from the view-point of availability. The enzyme can be stored for a long time, used repeatedly and available at a concentration higher than the MIC of the original bacteria.
Representative bacteria or fungi for the acylase source include strains of microorganisms belonging to e.g. genera Acetobacter, Achromobacter, Aeromonas, Alkaligenes, Arthrobacter, Blevibacterium, Beneckea, Bacillus, Corynebacterium, Escherichia, Flavobacterium, Gluconobacter, Kluyvera, Microbacterium, Micrococcus, Nocardia, Proteus, Pseudomonas, Rhodopseudomonas, Spilirum, Staphylococcus, Xanthomonas, Aphanocladium or Cephalosporium, or natural or artificial mutants or variants thereof capable of producing acylase for the condensation of this invention. Such strains include those described in e.g. Advances in Applied Microbiology, Volume 20, page 217 (1976) or natural or artificial mutants thereof available in the reaction of this invention.